Mycobacteria Invasion and Persistence

描述(申请人提供)：分枝杆菌感染仍然是人类健康的主要问题。结核分枝杆菌感染了世界约三分之一的人口，麻风分枝杆菌(ML)是世界许多地区的地方性感染。海洋分枝杆菌是鱼类的一种天然病原体，可引起具有许多结核病特征的青蛙的慢性疾病。正因为如此，MM在体外生长迅速，适合于正向遗传学，对实验室工作人员的致病性最低，以及在哺乳动物巨噬细胞中体外生长的能力，MM可以作为一种简便的模型来发现参与分枝杆菌感染发病机制的基因。我们已经开发出一种用于MM的转座子诱变质粒，它已被证明提供了一个极好的随机突变文库。从这个随机插入文库中，我们筛选了1000个能够在巨噬细胞中生长的突变体，并鉴定了25个不能正常生长的突变体。对于每个突变体，我们已经对转座子插入位点进行了测序，并利用这些信息发现了MM细胞内生长所需的几个基因。我们建议将重点放在两个最有趣的突变体的更详细的特征上，以了解目标基因在分枝杆菌入侵宿主细胞和细胞内生长中的作用。在每种情况下，我们都证明了靶向mm基因的Mtb同源物将补充在突变体中发现的表型缺陷。这使得快速和彻底地研究这些结核分枝杆菌基因在一种能够在细胞内生长并最终扩展到体内感染模型中的作用。目前的建议的目的是利用这些突变体来更好地了解分枝杆菌进入巨噬细胞和在巨噬细胞中生存，以及巨噬细胞对分枝杆菌感染的反应，这是疾病发病机制中的关键事件。我们的具体目标是：1.确定巨噬细胞侵袭宿主细胞和细胞内存活所需的MIP基因。2.确定GDP-甘露糖合成操纵子如何调控巨噬细胞激活和细胞内存活。3.对巨噬细胞内生长所需的mm基因进行遗传筛选。

DESCRIPTION (provided by applicant): Mycobacteria infections remain a major problem in human health. Mycobacterium tuberculosis infects approximately 1/3 of the world's population; Mycobacterium leprae (ML) is an endemic infection in many parts of the world. M. marinum (Mm) is a natural pathogen of fish and can cause chronic disease in frogs with many features of tuberculosis. Because of this, its rapid growth in vitro, its suitability for forward genetics, its minimal pathogenicity for laboratory" workers, and its ability to grow in mammalian macrophages in vitro, Mm can be a facile model to discover genes involved in pathogenesis of Mycobacteria infections. We have developed a transposon mutagenesis plasmid for use in Mm that has proven to provide an excellent library of random mutations. From that random insertion library, we have screened >1000 mutants for ability to grow in macrophages and have characterized >25 mutants that fail to grow normally. For each mutant, we have sequenced the transposon insertion site, and, using this information, we have discovered several genes required for intracellular growth of Mm. We propose to focus on a more detailed characterization of two most interesting mutants to understand the roles of the targeted genes in host cell invasion by, and intracellular growth of, Mycobacteria. In each case, we have shown that the Mtb homologue of the targeted Mm genes will complement the phenotypic defects discovered in the mutant. This allows a rapid and thorough investigation of the roles of these Mtb genes in a model that is capable of intracellular growth and ultimately of extension to in vivo models of infection. The purpose of the current proposal is to use these mutants to develop a better understanding of Mycobacteria entry into and survival in macrophages, and of macrophage response to Mycobacteria infection, critical events in the pathogenesis of disease. Our specific aims are: 1. Characterize the mip locus, required by Mm for host cell invasion and intracellular survival in macrophages. 2. Determine how the GDP-mannose synthesis operon regulates macrophage activation and intracellular survival. 3. Carry out a genetic screen for Mm genes specifically required for intracellular growth in macrophages.